Method and apparatus for identifying chromosomes or cells

ABSTRACT

A method and an apparatus for sorting chromosomes or cells by utilizing a difference in fluorescent profilepatterns. A linear laser beam having a high beam intensity is formed from several fringe patterns, and fluorescence intensities from local portions of chromosome or cell are measured in real-time. Resolution of chromosome or cell is improved. A type of flowing chromosome or cell which permits the rotational conditions within a range can be sorted accurately and rapidly.

This is a continuation of application Ser. No. 161,064 filed Feb. 26,1988 now U.S. Pat. No. 5,041,733..

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus and a method in whichdifferences in fluorescence intensity at narrow strips of local portionsof individual chromosome or cell flowing through a flow chamber aremeasured, so that chromosomes or cells of a specific type are sorted andcollected on the basis of patterns of the differences.

2. Description of the Prior Art

In order to classify chromosomes or cells of a specific type, variousmethods have been used in the fields of medical science, biology and thelike.

Heretofore, such a method in which chromosomes are classified inaccordance with parameters relating to the shape of a chromosome such asa size of the chromosome, e.g., a length of a long-arm portion or ashort-arm portion of the chromosome, centrometric index and the like onthe basis of the image by an optical microscope has been adopted as amethod for classifying chromosomes. However, such a method in which achromosome in a cell is stained with quinacrine mustard, and a type ofchromosomes is identified on the basis of a difference in patterns offluorescent lateral fringes (Q-band) which indicate characteristicfeatures of the chromosome was discovered by T. Casperson in Sweden in1970. A method for staining chromosome according to the above describedmethod is called as Q-differential staining. Other than thisQ-differential staining, C-differential staining and G-differentialstaining were discovered and by which a type of chromosome is classifiedon the basis of a difference in lateral fringes of a higher fluorescenceintensity which are called as C-band and G-band differing from Q-band.As described above, at present various chromosome types can beaccurately and fully identified on the basis of a difference influorescent band patterns appearing on a chromosome.

In order to identify gene of eucaryote, a method for making clear genesof individual chromosomes is employed by the properties of genescontained in the chromosome of a respective type. For this purpose, itis required to collect only a large amount of gene of a specific type inliving conditions. Furthermore, in the fields of tumorlogy andimmunology, it becomes necessary to collect a large amount of only cellsof a specific type such as subgroup of lymphocyte in living conditions.

As a method for classifying cells, there are a method in whichmorphologic characteristic features of cells or differences in physicalproperties of cells are skillfully utilized, and a method in whichdifferences in surface membrane of cells are utilized Moreover, such amethod in which the surface of a cell is labelled with a specificfluorescent material to identify a variant of the cell by means offluorescence microscope has been effected.

In addition to the methods as described above, such a manner that adevice called as a cell sorter begins to spread with rapidity. In thecell sorter, cells or chromosomes which have been previously labelledwith a fluorescent material, flow through a flow chamber or in a jetstream from a flow chamber nozzle at a high-speed. These cells orchromosomes are irradiated with a laser beam, and a fluorescentintensities of the chromosomes or cells as a result of the irradiationis measured, and the measured fluorescent intensities are analyzed,whereby specific chromosomes or cells are collected and sorted. Thismethod is of a special importance in the fields of cytochemistry,immunology, tumorlogy, genetics, molecular biology and the like.

Nevertheless, in a conventional cell sorter, cells or chromosomes havebeen irradiated with a laser beam having a far larger section than thatof a size of the cells or chromosomes flowing through a tube-shapedaqueous solution, so that the intensity of fluorescence emitted from thecells or chromosomes which had been previously labelled as a result ofthe irradiation has been measured Therefore, a conventional apparatus iscalled as a zero-resolution apparatus. In these circumstances, thedifferences in fluorescence intensity derived from local points of cellsor chromosomes could not be identified. For this reason, cells orchromosomes of a limited specific type could not be sorted andcollected.

For instance, a method which is utilizing a dual beam apparatus has beenproposed by P. N. Dean et al. ("High Resolution Dual Laser FlowCytometry", J. Histochem. Cytochem., Vol. 26, pp. 622-627, 1978.) Inthis method, LLL 761 chromosomes derived from human pellicle cell arebroken up into pieces, then these are stained with fluorescent materialsof Hoechst 3325Z and Chromomycin A3, and thus the stained pieces areirradiated with two laser beams of ultraviolet light and visible light,whereby fourteen types of chromosomes are uniquely identified inaccordance with such high resolution analysis. However, human ninth totwelfth chromosomes could not be sorted and collected by means of aconventional cell sorter and even the dual beam apparatus.

Then, R. V. Lebo et al., indicated that the human ninth chromosome canbe separated from a group of the human tenth to twelveth choromosomes bylabelling the human chromosomes with two kinds dyes of DIPI andchromomycin (R. V. Lebo et al., "Science," Vol. 225, 6 Jul., 1984, pp.57-59). However, the human tenth to twelfth chromosomes can not beseparated and collected by means of a conventional cell sorter.

As a method for improving a resolution power of a conventional flowcytometer, it is important how is a laser beam with which chromosomes orcells are to be irradiated formed. L. L. Wheeless et al., and Cambier etal., have developed the apparatuses in which the maximum radius of cellnucleus of a flowing cell and the size of the cell are measured. (L. L.Wheeless: "Slit-Scanning and Pulse Width Analysis", pp. 125-135, in FlowCytometry and Sorting, John Wiley and Sons, 1979. J. L. Cambier et al.:"A Multidimensional Slit-Scan Flow System", J. Histochem. Cytochem., pp.321-324, 1979)

However, in the above-mentioned apparatuses, since the slit width iswide, when a chromosome or cell passes through the flow chamber thevariation of the size of chromosome or cell with time is not measuredevery moment, but only the maximum radius of chromosome or cell at thetime of passage is measured.

On the other hand, a research group in the Lawrence Livermore NationalLaboratory has developed a method for narrowing laser beam in which twolaser beams are interefered and the obtained fringe-pattern is used(Norgren et al.: "Resoration of Profiles from Slit-Scan Flow Cytometry",IEEE Transactions on Biomedical Engineering, Vol. BME-29, pp. 101-106(1982)). For example, according to the Young interference method and theMach-Zehnder interference method, a vertical fringe pattern can beformed. Furthermore, a ring-shaped fringe pattern can be formed inaccordance with the Fabry-Perot interference method.

A method in which flowing chromosomes at a high-speed are irradiatedwith a fringe pattern formed in accordance with the Mach-Zehnderinterference method is known by Richard M. Norgren et al., U.S. Pat. No.4,596,036 "Method and Apparatus for Fringe-Scanning ChromosomeAnalysis".

In the method of R. M. Norgren et al., however, since flowingchromosomes are irradiated with laser beams of a pattern having severalfringes and a high beam intensity, the measurement of such fluorescenceintensity emitted by several laser beams of vertical fringes means tomeasure the total amount of the fluorescence intensity. Accordingly, itis concluded that the fluorescence intensity emitted from the localportions of the vertical fringe of chromosome can not be obtained so faras data as to the total amount of fluorescence intensity are analyzed.For this reason, it was impossible to measure a fluorescent bandpattern, a length and the like of chromosome in real-time in accordancewith conventional methods.

In this connection, a development for an apparatus and a method forsorting and collecting a specific cell or chromosome while maintainingbiological activity on the basis of differences in local fluorescenceintensity of chromosome or cell has been strongly desired.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an apparatus by which afluorescent band pattern of flowing chromosomes or a length ofchromosome can be measured in real-time.

Another object of the present invention is to provide an apparatus bywhich differences in fluorescence intensity derived from tens of localportions of individual cell or chromosome can be identified.

A further object of the present invention is to provide a method inwhich differences of the fluorescence intensity in band patterns derivedfrom local portions of flowing chromosomes or cells are identified, andchromosomes or cells of a specific type which could not have beenheretofore sorted are collected.

In the first aspect of the present invention, an apparatus foridentifying a chromosome or cell comprises:

a means for flowing an aqueous suspension containing chromosomes orcells which have been previously labelled with fluorescent materials;

an irradiation means provided with a laser beam source, an interferencemeans wherein a part of laser beams emitted from the laser beam sourceis branched, and the laser beam branched is combined again with theemitted beams to form laser beams having a fringe pattern of lateralfringe perpendicular to a flow axis along which the chromosomes or cellsflow, an optical means converting interference laser beams having thefringe pattern into elliptical beams in which a direction of the flowaxis is flat, and a slit for allowing only one interference fringecontained in the interference beams to pass therethrough to irradiatethe chromosomes or cells with the laser beam containing only the singleinterference fringe;

a fluorescence measuring means for measuring an intensity offluorescence emitted from the chromosomes or cells due to theirradiation; and

an identifying means for detecting the signal output from thefluorescence measuring means to identify chromosome or cell of aspecific type.

Here, a dimension of the slit in the flow axis direction may be 0.6 to1.5 μm with respect to the direction of the flow axis.

A breadth of the slit may be 50 to 300 μm.

The irradiation means may be provided with two laser beam sources.

The laser beams forming a crossing angle of 45° with each other in aplane perpendicular to the flow axis may be emitted from the two laserbeam sources.

In the second aspect of the present invention, an apparatus foridentifying a chromosome or cell comprises:

a means for flowing a liquid containing chromosomes or cells at ahigh-speed;

a means for emitting light onto the chromosomes or cells flowing throughthe flowing means;

a fluorescence measuring means for measuring fluorescence-intensityemitted from a fluorescent material with which the chromosomes or cellshave been previously labelled;

a discriminating means wherein a fluorescent profile-pattern obtainedfrom variation of the fluorescent intensity with time is compared with aplurality of the preset standard fluorescent profile-patterns ofchromosome or cell; and

a sorting means for sorting the chromosomes or cells on the basis of thecomparison of the fluorescent profile-pattern with the preset standardfluorescent profile-patterns by the discriminating means.

Here, the standard profile-pattern may include a profile-patternrelating to a rotating condition of chromosomes or cells.

In the third aspect of the present invention, a method for identifying achromosome or cell comprises the steps of:

irradiating a flow-stream containing chromosomes or cells which havebeen previously labelled with a fluorescent material with a laser beamof only one interference fringe among interference laser beams having afringe pattern perpendicular to a flow axis along which the chromosomesor cells flow;

measuring fluorescence intensity emitted from the fluorescent materialon the chromosomes or cells;

comparing a fluorescent profile-pattern obtained from variation of thefluorescent intensity with time with preset standard fluorescentprofile-patterns of chromosome or cell; and

sorting the chromosomes or cells on the basis of the comparison of thefluorescent profile-pattern with the preset standard fluorescentprofile-patterns.

The above and other objects, effects, features and advantages of thepresent invention will become more apparent from the followingdescription of preferred embodiments thereof taken in conjunction withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanatory diagram showing an embodiment of the presentinvention;

FIG. 2 is a perspective view showing an optical system which generates alaser beam and is used in the embodiment of the present invention inFIG. 1;

FIG. 3 is a schematic view showing the flow chamber and the fluorescencemeasuring system in FIG. 1;

FIGS. 4A and 4B are explanatory diagrams showing a fringe pattern of alaser beam and an intensity distribution of an interference laser beam,respectively;

FIG. 5 is an explanatory diagram illustrating a method for generating alinear laser beam;

FIG. 6 is a graphical representation illustrating an intensitydistribution of a diffracted beam due to a rectilinear slit;

FIGS. 7A and 7B are explanatory diagrams showing a band pattern of achromosome and a fluorescent profile-pattern, respectively;

FIG. 8 is a block diagram showing a schematic constructional example ofan electronic circuit for analyzing a fluorescent profile-pattern;

FIG. 9 is an explanatory diagram illustrating a relationship betweenEulerian angles and rotational coordinates axes; and

FIG. 10 is a diagram showing calculated results of computer simulationin respect of fluorescent profile-patterns of chromosome.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 illustrates an outline of an embodiment according to the presentinvention. This apparatus comprises a) a laser beam generating andshape-forming portion, b) a sample flowing portion, c) a water dropletsorting portion, d) a fluorescence detecting portion, e) a high-speedprofile-pattern processor, and f) a central processing unit. Therespective portions a) to f) of the present apparatus will be describedin more detail hereinbelow.

The laser beam generating and shape-forming portions are composed of twosets of laser beam oscillators 2A and 2B for emitting laser beam 1A ofultraviolet rays and laser beam 1B of visible rays, respectively, andtwo sets of optical systems 3A and 3B such as lenses for forming a laserbeam with traverse section being a submicron in thickness and severalhundreds μm in width, respectively. In the above construction, when twosets of the laser oscillators 2A and 2B are employed, laser beams havingdifferent wavelengths can be used as light sources, so that it ispossible that two kinds of DNA specific dyes can be combined forlabelling chromosome or cell.

The sample flowing portion b) is composed of a flow chamber 6 forchanging a flow stream 5 containing chromosomes or cells 4 intosheath-like laminar flow, a sheath liquid 7 for positioning the flowstream 5 at the center of the flow, a pressure device 8 for maintaininga high speed of the flow, and a piezoelectric vibrator 9 for affording aminute vibration to the flow chamber 6. When the piezoelectric vibrator9 is minutely vibrated, droplets are formed in every second coincidentlywith the frequency of the piezoelectric vibrator.

The water droplet sorting portion c) is composed of a droplet chargingunit 11 for applying positive or negative electric charge to a droplet10 containing one cell or chromosome, deflection plates 12 forseparating droplets by deflecting the falling droplets 10 from thedirection along which the droplets drop by means of a force of electricfield, a droplet capturing device 13 for capturing the droplets 10 whichhave been deflected by the deflection plates 12, and a container 14 forundeflected droplets which collects waste liquid: In order to chargedroplets, weak electric current is applied between an electrode which isin contact with the flow stream at the position of a nozzle in the flowchamber and a ring-shaped electrode encompassing the droplets 10. Anapparatus described herein is the same as a conventional cell sorterexcept the optical systems 3A and 3B. (L. A. Herzenberg et al.:"Fluorescence-activated cell sorting", Scientific American, pp. 108-117,Mar., 1976).

The fluorescence detecting portion d) is composed of lenses 16A and 16Bfor collecting fluorescences 15A and 15B emitted from labelledchromosomes or cells with a fluorescent material, dichroic filters 17Aand 17B for dividing the collected fluorescence into spectra of twowavelength bands, and several photomultipliers 18A-18D for detecting thefluorescence and converting the results detected into current.

The high-speed profile-pattern processor 19 contains a circuit forshaping and amplifying an output pulse waveform from the photomultiplier18, a circuit for sampling the pulse waveforms, an A/D converter fordigitalizing a value of voltage sampled, a mass storage element, and acomparator.

The central processing unit 20 contains a microcomputer, a cathode raytube display, a printer, and a keyboard. Furthermore, a large auxiliarystorage 21 is connected to the central processing unit 20.

It is to be noted that a temporal disperser photometer 22 is a devicefor measuring a rough two-dimensional shape of flowing chromosome orcell.

The operation of the present embodiment having the construction shown inFIG. 1 will be described hereinbelow.

The sheath liquid 7 made of physiological saline is fed to the flowchamber 6 by means of the pressure device 8, and at the same time thesample liquid 5 in which an object to be detected, that is, chromosomeor cell 4, floats is fed into the flow chamber 6 at a position of thecentral axis so as to wrap the sample liquid in the sheath liquid. Inthis case, the cells or chromosomes being the object to be detected havebeen previously stained with a fluorochrome material. In order tomaintain the cells at the center, a flow ratio between the sheath liquidand the sample liquid is suitably selected.

The flow chamber 6 is minutely vibrated by the use of the piezoelectricvibrator 9, and a droplet containing a cell or a chromosome is allowedto drop. The aqueous suspension flowing in the nozzle portions isirradiated with such laser beams 1A and 1B focussed by means of anoptical system which will be described in detail in FIG. 2. Thefluorescence emitted from the fluorescent material in the chromosomes orcells, which have been previously labelled, is collected by the lenses16A and 16B, the fluorescence thus collected is separated by thedichroic filters 17A and 17B into its spectral components, and anoptical signal is detected by means of the photomultipliers 18A-18D toconvert it into an electric signal. A detected signal from thephotomultipliers 18A-18D is analyzed by utilizing the high-speedprofile-pattern processor 19, the central processing unit 20, and thelarge auxiliary storage 21 so as to output such a signal whether or notthe detected cell or chromosome should be sorted from the centralprocessing unit 20.

When the signal from the central processing unit 20 is a sorting signal,electric charge is applied to the droplet 10 by means of the dropletcharging unit 11, and the droplet is attracted towards such deflectionplate 12 having the sign of charge which is opposite to that given tothe droplet 10 when the droplet passes through the deflection plates 12,whereby droplets containing chromosome or cell of a specific type areselectively collected in the water droplet capturing device 13. Dropletswhich should not be sorted are collected in the container 14 forundeflected droplets.

Next, a manner how a focussed laser beam is formed will be described indetail hereinbelow.

FIG. 2 is a view showing details of the optical system for forming laserbeam according to an embodiment of the present invention whereinreference numeral 201 designates a laser oscillator emitting continuousvisible radiation or ultraviolet radiation, the diameter of a laser beamis about 1.6 mm, 202 a beam splitter (half mirror), 203 and 204 mirrors,205 a beam splitter (half mirror), 206 a cylindrical lens, 207 a shadehaving a slit 207A of several hundreds μm in breadth and about 1 μm inwidth, and 208 the flow chamber shown in FIG. 1 which forms a sampleliquid containing cells or chromosomes into a sheath-like laminar flow,respectively. Chromosomes or cells pass through the flow chamber 208along the central axis thereof. An external form of a nozzle portion ofthe flow chamber 208 is a regular octagon. In this case, a diameter of apore defined on the nozzle portion for permitting cells or chromosomesto pass therethrough one by one is about 200 μm. In the presentembodiment, since the cells or chromosomes passing through the pore ofthe nozzle portion are irradiated with a laser beam, a materialconstructing the nozzle portion is selected from materials, for example,synthetic quartz through which the laser beam can pass uniformly. Inthis case, since an oil or glycerine for oil immersion is applied to agap between the regular octagonal outer wall of the nozzle portion andthe slit 207, the outer wall of the nozzle portion is in close contactwith the slit. Furthermore, reference numeral 209 designates anobjective lens, 210 an interference filter such as a long pass filter,211 a convex lens, 212 a pinhole, and 213 a dichroic filter,respectively.

Next, irradiation of laser beam in the optical system shown in FIG. 2 aswell as emission of fluorescence will be described in detailhereinbelow.

First of all, a laser beam 214 is emitted from the laser oscillator 201.In this case, a direction along which the laser beam 214 proceeds is setto be the X-axis. The Y-axis is in a direction along the centerline ofan axis through which cells or chromosomes flow at a high-speed in theflow chamber 208. The regular direction of the Y-axis is opposite tothat along which the chromosomes or cells flow. The Z-axis is determinedin a right-hand system composed of the X-, Y-, and Z-axis. The laserbeam 214 emitted along the regular direction of the X-axis is dividedinto a passing laser beam 215 and a reflected laser beam 216 by means ofthe beam splitter 202. The reflected laser beam 216 is reflected by themirror 203 to form a laser beam 217, and the laser beam 217 is furtherreflected by the mirror 204 to form a laser beam 218. The passing laserbeam 215 is in parallel to the reflected laser beam 217.

The passing laser beam 215 passes through a neutral filter 225 betweenthe beam splitters 202 and 205 to form a laser beam 219. The neutralfilter 215 is a filter for adjusting a beam intensity of the laser beam218 so as to be equal to that of the laser beam 219 in the beam splitter205. In this situation, if the beam intensity in these two laser beamsis not equal to each other, contrast in beam intensity in crest andtrough of a fringe pattern as mentioned hereinbelow becomes weak. In thebeam splitter 205, the laser beam 218 interferes with the laser beam 219to form a laser beam 202 of a pattern of several fringes.

Such a method in which an optical system is composed of the beamsplitters 202 and 205 as well as the mirrors 203 and 204, and two laserbeams 218 and 219 are allowed to interfere with each other is generallycalled as the Mach-Zehnder interference method. The optical axes of thebeam splitters 202 and 205 intersect the optical axes of the mirrors 203and 204 to form a parallelogram.

An extent of a laser beam 220 in the direction of the Y-axis is reducedby means of the cylindrical lens 206, so that it becomes a laser beam221 having a high beam intensity distribution along the direction of theZ-axis. The laser beam 221 passes through a slit 207A, so that ittransforms from a laser beam of a high intensity having a pattern ofseveral fringes into a laser beam having a pattern of only one fringewith high intensity. Such chromosomes or cells flowing along the centralaxis of the flow chamber 208 are irradiated with the laser beam having apattern of substantially only one fringe and light 222 containinglateral scattered light and fluorescence is emitted. The emitted light222 is collected by means of the objective lens 209. The fluorescencepasses through the interference filter 210, while the lateral scatteredlight does not pass through the interference filter 210.

The fluorescence appears on the side of a longer wavelength than that ofthe laser beam for excitation. When the fluorescence is focussed by theconvex lens 211 and allowed to pass through the pinhole 212, only thefluorescence emitted from the chromosomes or cells is obtained, whileoutwardly scattered light and noisy light are interrupted by means ofthe pinhole 212. The latter fluorescence is further spectrally separatedby the use of the dichroic filter 213 into the one having a specifiedwavelength band.

FIG. 3 is a constructional view of a flow chamber and a fluorescencephotometric system wherein the X- and Z-axis have the same meaning asthose appeared in the description of FIG. 2. Reference numerals 306A and306B designate cylindrical lenses, and 307A and 307B shades each havingan opening, respectively. A dimension of the opening is the same size asthat of the slit 207A shown in FIG. 2. A flow stream containingchromosomes or cells flows at a high-speed through a pore 326, and adiameter of the pore 326 has already been described in the descriptionas to the nozzle portion of FIG. 2. A material and an outer shape of anozzle portion 327 in the flow chamber are the same as those of the flowchamber 208 shown in FIG. 2. The focal points of objective lenses 309Aand 309B coincide with the central axis of the pore 326 in the flowchamber. Interference filters 310A and 310B are allowed to pass onlyfluorescence therethrough. The focal points of convex lenses coincidewith the positions of pinholes 312A and 312B, respectively. The pinholes312A and 312B function to pass through only fluorescence emitted fromchromosomes or cells.

Next, irradiation of laser beam in the optical system shown in FIG. 3and emission of fluorescence will be described hereinbelow.

In FIG. 3, chromosomes or cells are irradiated with two laser beams 320Aand 320B having different wavelengths from each other. This is becausetwo or more fluorescent probes are employed. For the simplicity, it isassumed that reference numeral 320A designates a laser beam ofultraviolet rays, and reference numeral 320B designates a laser beamhaving a wavelength of a visible region. The laser beam 320A proceeds tothe central axis of the pore 326 along the direction of the X-axis,while the laser beam 320B proceeds to the central axis of the pore 326at an angle of 45° defined by the laser beam 320B and the X-axis. Inthis case, the laser beam 320A is in the same plane as that of the laserbeam 320B. After the laser beams 320A and 320B having a pattern ofseveral fringes formed by interference of these laser beams passedthrough the cylindrical lenses 306A and 306B, laser beams with severalfringes have high beam intensities in horizontal direction such as thinthickness beam. Furthermore, after these laser beams passed through theshades 307A and 307B each having a slit, the intensity of one centralfringe beam becomes much higher than those of the other fringe beams.The laser beam having a thin thickness in intensity which has passedthrough a slit is hereinafter referred to as "linear laser beam".

When chromosomes or cells flowing through the pore 326 opened along thecentral axis of the nozzle portion 327 of the flow chamber areirradiated with two laser beams, forward scattered light as well asfluorescence 318A and 318B are emitted.

In this case, such beam emitted as a result of the irradiation of cellsor chromosomes with laser beam is observed in the rectangular direction.This is because fluorescence to be measured appears on the side of alonger wavelength than that of excited light due to irradiation withlaser beam and further its light intensity is weak. Emitting beams 318Aand 318B from the cells or chromosomes become parallel beams when thebeams pass through the lenses 309A and 309B. When these two beams passthrough the interference filters 310A and 310B, there remains onlyfluorescence. When two fluorescent beams pass through the convex lenses311A and 311B, they are converged and pass through the pinholes 312A and312B. Thereafter, the fluorescence is spectrally separated by thedichroic filter, the separated fluorescence is detected by means of thephotomultiplier, and the signal detected is analyzed to sort specificchromosomes or cells.

FIGS. 4A and 4B show a fringe pattern of laser beam and an intensitydistribution of the laser beam at traverse section wherein FIG. 4A is anenlarged view showing a vertical stripe-like fringe pattern appeared ina circle having a diameter of about 1.3 mm which exhibits an outerboundary of an extent where the laser beam emitted from a laser beamsource extends. In FIG. 4A, the portions darkened in the circle indicateportions where a beam intensity is high, the direction of a verticalfringe corresponds to that of the Z-axis, and the perpendiculardirection to the vertical fringes is considered to be that of theY-axis, i.e., the direction of a flowing axis along which chromosomes orcells flow.

FIG. 4B illustrates a beam intensity distribution of the laser beam inthe direction of the Y-axis, the laser beam having the fringe patternshown in FIG. 4A. From FIG. 4B, it is found that several linear laserbeams having a high beam intensity in the direction of the X-axisproceed in parallel to each other.

FIG. 5 is an explanatory view for a method for forming a linear laserbeam having substantially one high beam intensity and a thin thicknessfrom a laser beam having a pattern of several fringes.

The method for forming a linear laser beam relates to the one in which alaser beam having a pattern of several fringes is allowed to passthrough a substantially linear slit.

In this embodiment, a shade having a slit is composed of a slitsubstrate 529 made of synthetic quartz having a thickness of about 300μm and a chromium coating layer 528 having a thickness of about 0.1 μmwhich has coated the slit substrate 529. The slit through which a laserbeam 521 passes is opened in accordance with a publicly knownphoto-etching technique, so that an opening having a thickness(thickness in the Y-axis direction) of 1 μm and a breadth (length in theZ-axis direction) of about several hundreds μm is obtained. In thiscase, a thickness of the slit is substantially determined by ahalf-value width of the fringe pattern and a wavelength of the laserbeam, and it may be determined within a range of 0.6-1.5 μm.Furthermore, in order that a flux portion of a flow chamber throughwhich chromosomes or cells flow is easily irradiated with linear laserbeam, a breadth in the slit is defined wider than a diameter of a pore(50-200 μm) in the nozzle portion of the flow chamber, and it is usuallywithin a range of 50-300 μm.

A distance between the shade having a slit and the inner wall of thenozzle portion of the flow chamber is determined to be 1-3 mm. In thiscase, a laser beam 521 which has passed through a cylindrical lens topossess a fringe pattern of the beam intensity in its breadth directionhas a beam intensity distribution represented by a dotted curve 527. Inthese circumstances, when the laser beam 521 which has proceeded in thedirection of the X-axis passes through a slit having a thickness of 1μm, only such a linear laser beam having a pattern of substantiallysingle fringe of a highest beam intensity remains among laser beams eachhaving a pattern of several fringes. Chromosomes or cells which flowthrough the pore 526 of the nozzle portion of the flow chamber at a highspeed along a flow axis 530 of the flow chamber are irradiated with thelaser beam which has passed through the aforesaid slit.

As a result, a thin laser beam having a thickness of a submicron orderis formed. In this case, it is required for forming a linear laser beamwhose intensity is high that accuracy of a cylindrical lens and abreadth of the slit have been sufficiently adjusted to correct.According to the manner as described above, it is possible to narrowdown a thickness of such a linear laser beam up to about half thewavelength of a laser beam.

A problem in the manner of the present embodiment is in that a linearlaser beam emitted from the linear slit is diffracted at an opening ofthe slit.

The case of the Fraunhofer diffraction which is observed in the eventwhere a distance between a slit and a laser beam source and between aslit and a point to be irradiated in the flow axis are comparativelylong will be considered with taking such fact that an opening of theslit is rectangular into consideration (for example, see Max Born & EmilWolf "Principles of Optics", Pergamon Press., 1975). In the case of theFraunhofer diffraction, a diffracted intensity I is generally given asfollows: ##EQU1## wherein u₁ and u₂ are dependent upon a thickness andbreadth of a slit, a wavelength of laser beam, and a distance from aslit to a point to be irradiated and to a laser beam source, but theydepend principally upon an angle of diffraction.

FIG. 6 shows an intensity distribution of diffracted light by means of arectilinear slit. If the slit is linear, one of u₁ and u₂ is 0, so thatthe diffracted intensity I in this case is given as follows: ##EQU2##wherein u is dependent upon a width of the slit, an angle ofdiffraction, a wavelength of laser beam, and a distance between a slitand the points to be irradiated and between a slit and a laser beamsource.

An intensity of diffracted light in the case where the slit is arectilinearly opened slit has such a distribution which is substantiallyproportional to square of the curve shown in FIG. 6.

From FIG. 6, it is understood that the laser beams are composed of alinear laser beam whose intensity is the highest in the horizontaldirection (the Z-axis) being perpendicular to that along which the laserbeam propagates and another laser fringe beams which are parallel to theformer laser beam and are very weak in intensity.

Accordingly, one laser beam having a wide breadth and a thin thicknessamong laser beams which proceed in the onward direction excites afluorescent material within the narrow strip of local portions inchromosomes or cells flowing in a flow chamber at a high-speed along theflow axis thereof. Since such another laser beam which is in parallel tothe laser beam having the highest beam intensity exhibits a weak beamintensity, it does not contribute to the excitation of fluorescentmaterial so much. Even if there is fluorescence emitted as a result ofirradiation of laser beam, such fluorescence becomes background noise.

Such a case where chromosomes are irradiated with the linear laser beamformed as mentioned above will be considered hereinbelow.

FIG. 7A is an explanatory view of chromosome showing a situation whereas an example, a chromosome having an entire length of 10 μm is allowedto flow through a nozzel diameter of 100 μm of a flow chamber at a flowvelocity of 1 m/sec., in the direction from the upper part to the lowerpart in FIG. 7A. In this figure, a hatched portion indicates a sectionof a linear laser beam having a thickness of 0.5 μm, and a indicates aband pattern of the labelled chromosome with a fluorescent material.

FIG. 7B is a graphical representation indicating a change with time inrespect of a fluorescent intensity emitted from the chromosome shown inFIG. 7A wherein the abscissa indicates a time elapsed from the beginningof irradiation of the chromosome with a linear laser beam, while theordinate indicates a relative fluorescent intensity to be detected, andin this case, since the entire length of the chromosome is 10 μm and theflow velocity is 1 m/sec., the elapsed time is 10 μsec.

Since the linear laser beam has a thickness of a submicron order, achromosome is excited at only a strip of submicron width. In this case,fluorescent luminous energy irradiated is proportional to an amount ofthe fluorescent material which is contained in a strip of thechromosome. The fluorescence detected in a photomultiplier is convertedinto an analog value which varies sequentially in a continuous manner,and then the value is output. This analog signal is converted into adigital value which can be represented in 8 or 16 bits by sampling theanalog signal at an interval of 500 nsec., with the use of adigital/analog (A/D) converter. In this case, an entire length ofchromosome is 10 μm and a width of liner laser beam is 0.5 μm, so thatthe resultant output of the A/D converter is represented by atwenty-dimensional vector with respect to individual chromosome or cell.While a flow velocity of chromosome and a sampling time intervaltherefore are considered to be 1 m/sec., and 500 nsec., respectively,for the sake of simplicity. An actual flow velocity of chromosome is farrapid, and a sampling time interval is far short. Accordingly, a seriesof the digital values obtained every moment becomes a tens-dimensionalvector in general. This tens-dimensional vector is reflective of localdifferences in fluorescence intensity of chromosome or cell. In thisconnection, such a tens-dimensional vector is referred to herein as"fluorescence profile-pattern".

FIG. 8 is a block diagram showing a constructional example of anelectronic circuit which identifies type of chromosome or cell byanalyzing a fluorescence profile-pattern wherein reference numerals 81Ato 81D designate photomultipliers (PMT), 82A to 82D amplifiers, 83A to83D sample and hold circuits (S/H), 84A to 84D analog-digital convertingcircuits (A/D), 85 a high-speed memory, 86 a memory bank, 87 ahigh-speed I/0 bus, 88 a memory for weight coefficient pattern, 89 amemory for sort pattern, 90 a microcomputer, 91 an interface, 92 apattern matching circuit, 93 a standard profile-pattern generator, and94 a weight coefficient generator, respectively.

Next, operation of the electronic circuit shown in the block diagram ofFIG. 8 will be described hereinbelow.

Emitting fluorescent beams A and B obtained in the case when chromosomesor cells are irradiated with ultraviolet laser are detected by the PMT81A and 81B, respectively. On the other hand, emitting fluorescent beamsC and D when chromosomes or cells are irradiated with a visible lightlaser beam having a different wavelength with that of theabove-mentioned ultraviolet laser beam are detected by the PMT 81C and81D, respectively. In this case, the PMT 81A to 81D are selected fromones suitable for a wavelength region of fluorescent beams A to D.Electric signals output from the PMT 81A to 81D are subjected towaveform shaping and amplified by means of the amplifiers 82A to 82D.Analog voltage signals from the amplifiers 82A to 82D are sampled bymeans of the S/H 83A to 83D at a time interval of from several tensnanoseconds to several hundreds nanoseconds to hold the resultingsampling values. In the A/D converters 84A to 84D, the voltage valueswhich have been sampled and held are converted into digital values of 8or 16 bits.

The tens-dimensional fluorescent profile-patterns thus obtained anddigitalized are transferred to the high-speed memory 85. Suchfluorescent profile-patterns of chromosomes or cells flowed in oncesample-flow are stored in the memory bank 86. The memory bank 86 is aset of memory elements having a capacity sufficient for storingfluorescent profile-patterns, and a memory capacity of the memory bankis around several megabytes to several tens megabytes. Informationstored in the memory bank 86 is properly output to the outside throughthe high-speed I/O bus 87.

On one hand, the microcomputer 90 calls a set of standardprofile-patterns necessary for identifying a specific type of chromosomeor cell from the standard fluorescent profile-patterns generator 93 tostore the standard profile-pattern in the memory 89 for sort patternthrough the interface 91. Furthermore, the microcomputer 91 calls a setof weight coefficients from the weight coefficient generator 94 to storethe weight coefficients in the memory 88 for weight coefficient patternthrough the interface 91.

Then, in the pattern matching circuit 92, such a standardprofile-patterns which have been multiplied by weight coefficients arecompared with an input fluorescent profile-pattern from the high-speedmemory 85 to identify the type of chromosome or cell, so that a sortsignal is output from the pattern matching circuit 92 in only the casewhen one of the results compared as described above exceeds a thresholdindicating a degree of coincidence of the patterns.

In a conventional cell sorter while identifying cells to be selected andcollected in a real-time, identification has been performed on the basisof such result whether or not a height of a voltage pulse which isreflective of the total fluorescence intensity produced at the time whensingle cell traverses a laser beam is within a preset voltage level. Inthe apparatus of the present invention, the identifying method is basedon a fluorescent profile-pattern of a tens-dimensional vector. However,since it is necessary to sort chromosomes or cells flowing at a highspeed, a period of time being capable of allotting for identifying atype of chromosomes or cells in the present apparatus is only severaltens microseconds. Accordingly, a comparatively simple procedure isutilized for identifying such a type.

In the present embodiment, since a width of laser beam is narrow, it maybe considered that a fluorescent profile-pattern is influenced by notonly the amount of fluorescent material contained in the narrow strip oflocal portions, but also an inclination of chromosomes or cells flowing.

For this reason, such standard profile-patterns in the case wherechromosomes or cells flow with several inclinations are previouslyestimated and they are preset at the pattern matching circuit. If suchprofile-patterns of chromosomes with slightly inclinated have not beenprepared, there is a possibility of missing chromosomes or cells to beselected and collected.

In order to obtain a standard profile-pattern of flowing chromosomes orcells with an inclination, the rotating state in the three dimensionalspace of chromosomes or cells is determined.

FIG. 9 is a graphical representation indicating a relationship betweenthe Eulerian angles and the rotational coordinate axes wherein adirection opposite to that along which a floating liquid containingchromosomes or cells flows is considered to be a positive direction ofthe z-axis, the x- and y-axis exist in a plane perpendicular to thez-axis, a direction opposite to that along which a laser beam propagatesis determined as the direction of the x-axis, and the y-axis directionis determined in such that the x-, y- and z-axis construct a right-handsystem. If origin of the rectangular coordinate axes is assumed to be apoint of intersection of a flow axis and the central axis of the laserbeam, the spatial coordinate axes may be expressed as (0; x, y, z). Whena substance rotates around the z-axis by an angle α radian, thecoordinates axes become (0; x₁, y₁, z). Then, when the substance rotatesaround the x₁ -axis by an angle β radian, the coordinates axes become(0; x₁, y₂, z₁). Furthermore, when the substance rotates around the z₁-axis by an angle γ radian, the coordinates axes become (0; x₂, y₃, z₁).These three angles (α, β, γ) are called as the Eulerian angles.

For example, the Eulerian angle (α, β, γ) are required for determining arotating state of flowing chromosomes. In this connection, since thechromosomes flow in the negative direction of the z-axis at a rate ofseveral meters per second, the longitudinal axial direction ofchromosome becomes a state where it extends substantially along thez-axis. Accordingly, an Eulerian angle which influences the mostsignificantly a variation of standard fluorescent profile-pattern isconsidered to be angle β. According to circumstances, there is such acase when a chromosome flows in a condition where the longitudinal axialdirection of the chromosome is inverted.

For this reason, standard fluorescent profile-patterns in the followingrotational conditions are prepared:

Eulerian angles (0°, 0°, 0°), (0°, 180°, 0°), (0°, 30°, 0°), (0°, -30°,0°), (0°, 150°, 0°), (0°, 210°, 0°), (0°, 30°, 30°), (0°, -30°, 30°),(0°, 150°, 30°), (0°, 210°, 30°), (0°, 150°, -30°) and (0°, 210°, -30°).Standard profile-patterns defined and specified by the above describedrotational conditions have been previously inputted into a standardprofile-pattern generator, and such standard profile-patterns aresuitably called out in the pattern matching circuit to compare them witha fluorescent profile-pattern inputted, thereby to identify chromosomesor cells to be sorted.

An example of results in computer simulation between a fluorescentprofile-pattern in the case where chromosomes flow in an inclinedposture and that of the case where chromosomes flow without anyinclination is illustrated in FIG. 10.

FIG. 10 indicates calculated results of an expected fluorescenceprofile-pattern in the case where it is assumed that chromosome is arectangular parallelepiped having a length of 9.0 μm, a breadth of 4.0μm and a thickness of 2.0 μm and that a thickness of a very thin linearlaser beam is 0.5 μm. In FIG. 10, the ordinate indicates the serialnumber of dimension indicating the number of times for measuringfluorescent intensities at different portions in one chromosome or cell.The abscissa indicates the relative fluorescent intensity, and positivesign "+" indicates a fluorescent profile-pattern in the case where thereis no rotational condition, i.e., an Eulerian angle is (0°, 0°, 0°). Theprofile-pattern indicates a fluorescent band pattern. A negative sign"-" bar graph indicates the fluorescent profile-pattern of therectangular parallelepiped with the fluorescent band pattern oriented atan Eulerian angle (20°, 10°, 20°). As described above, when a pluralityof standard fluorescent profile-patterns having different Eulerianangles are allowed to store in the apparatus according to the presentinvention, a type of cell or chromosome can be judged on the basis ofsuch a fact whether or not at least one of indices which indicates thecoincidence of the fluorescent profile-pattern with the standardfluorescent profile-patterns, exceeds a predetermined threshold.

Types of chromosome or cell differ from one another dependently upontypes of organism (for example, human being, hamster, bird, fish and thelike), and further fluorescent profile-patterns become different onesdue to differences in fluorescence staining methods or fluorescent DNAspecific dyes even in the same type of organism. Accordingly, there aresubstantially numerous standard profile-patterns of chromosome or cell.For this reason, it is impossible to always store all the standardfluorescent profile-patterns in the present apparatus.

However, types of organism to be identified, staining methods forfluorescent labelling, and in addition type numbers etc., of thechromosome or cell are known in advance in the case when a specific typeof chromosome or cell is to be selected and collected in the apparatusof the present invention. Moreover, since a type of chromosome or cellto be selected and collected relates to only one type, the chromosomesor cells of the type can be sorted by comparing input profile-patternsmeasured consecutively with the standard profile-patterns of the type,even if the chromosomes or cells are passing through with inclinatedpostures in limited angles by means of the apparatus according to thepresent invention.

As described above, when such standard profile-patterns are suitablycompared with a fluorescent profile-pattern inputted, a type of cell orchromosome to be sorted can be collected.

According to the present invention, only a fluorescence from a narrowstrip of local portions which has been previously labelled by afluorescent material can be measured in respect of a fluorescentintensity from such chromosomes or cells. Accordingly, there is no needfor analysis of the fluorescence intensity obtained from several fringepatterns, so that only fluorescence intensities from local portions ofindividual chromosome or cell are measured in real-time and they areimmediately utilized for sorting.

Furthermore, according to the present invention, chromosomes or cellsare locally irradiated with a laser beam whose thickness is far thinnerthan a diameter of a chromosome or cell, an intensity of fluorescenceemitted as a result of the irradiation is detected, and time sequentialsignals which are results of the detection are analyzed, so thatchromosomes or cells of a specific type can be sorted and collected.

As described above, chromosomes or cells having different characters andfunctions can be analyzed and separated, so that changes in quantity orquality of such chromosomes or cells can be measured quantitatively andrapidly in accordance with the present invention. Therefore, the presentinvention can significantly contribute to various fields such as medicalscience, biology and the like.

What is claimed is:
 1. A method for identifying a chromosome or cellcomprising the steps of:irradiating a flow-stream containing chromosomesor cells which have been previously labelled with a fluorescent materialwith a laser beam of only one interference fringe among interferencelaser beams having a fringe pattern perpendicular to a flow axis alongwhich said chromosomes or cells flow; measuring fluorescence intensityemitted from the fluorescent material on said chromosomes or cells;comparing a fluorescent profile-pattern obtained from variation of saidfluorescent intensity with time with preset standard fluorescentprofile-patterns of chromosome or cell; and sorting said chromosomes orcells on the basis of the comparison of said fluorescent profile-patternwith said preset standard fluorescent profile-patterns.